Patent UA-89249-C2 (UA)
Title: YEAST STRAIN PASTORIS PS107(pPIC9HAbIL-2), WHICH IS THE PRODUCER OF HYBRID PROTEIN, CONSISTING OF ALBUMIN OF HUMAN PLASMA AND HUMAN INTERLEUKIN-2, RECOMBINANT PLASMID pPIC9HAbIL-2 AND THE METHOD FOR DESIGNING THEREOF
Inventor: PADKINA MARINA VLADIMIROVNA
IPC: C12N1/19
Filed: 2008-02-06
Abstract: The invention relates to a pPIC9HAblL-2 recombinant plasmid, providing for biosynthesis and release of hybrid protein, which consists of albumin of human plasma and human IL-2, by the yeast cells, transformed by it, having the size of 10148 b.p., and which consists of the following elements: 1) EcoR1-Xho1 - fragment of plasmid DNA bifunctional bacterial-yeast vector pPIC9 with the size of 8.00 kb., bonded with restriction sites EcoR1-Xho1, and which comprises bacterial ampicillin resistance gene; bacterial region of inintiation of replication; yeast gene HIS4; fragment of 5'-noncoding region of yeast gene AOX1 having the size of 0.95 kb, which contains region, providing for activization of trenscription of this gene in the presence of methanol as a carbon source in cultural medium; preproregion of gene MF EMBED Equation.3 of yeast Saccharomyces cerevisiae having the size of 0.27 kb, which provides for release of hybrid protein, which consists of albumin of human plasma and human IL-2, into cultural medium; fragment of gene AOX1, having the size of 0.33 kb, containing the region of termination of transcription of this gene; and the fragment of 3'- noncoding region of gene AOX1 having the size of 0.76 kb; 2) Xho1-Bsu36l –fragment, having the size of 1749 b.p., containing the coding part of gene of albumin of human plasma with the exception of the region coding signal peptide, and stop codon; 3) Bsu36l-EcoR1 -, having the size of 399 b.p., containing the coding part of gene of human IL-2 with the exception of the region coding signal peptide. Also the invention relates to the yeast strain Pichia pastoris PS107(pPIC9HAblL-2, which is the producer of the mentioned hybrid protein, and which is the strain of Pichia pastoris PS99 (his4 pep4::PHO85), transformed by the mentioned plasmid. Also the invention relates to the method for designing of the mentioned recombinant plasmid pPIC9HAblL-2, which comprises a) gene of albumin of human plasma with the exception of the region coding signal peptide, and stop codon, is obtained by means of reverse PCL using of mRNA array, obtained from human hepatocytes, direct primer SEQ ID NO: 1, containing the site for restriction nuclease Xho1, reverse primer SEQ ID NO: 2, containing the site for restriction nuclease Mstll; b) gene of human IL-2 with the exception of the region coding signal peptide, obtained by means of PCR using of plasmid pJDB(MSIL) array, direct primer SEQ ID NO: 3, containing the site for restriction nuclease Bsu36l, reverse primer SEQ ID NO: 4, containing the site for restriction nuclease EcoR1; c) the obtained gene of albumin of human plasma is treated by restriction nucleases Xho1 and Mstll; d) the obtained gene of human IL-2 is treated by restriction nucleases Bsu36l and EcoR1; the genes of albumin of human plasma and human IL-2 are joined by ligase, and treated, as mentioned above, with plasmid pPIC9, preliminary treated by the restriction nucleases Xho1 and EcoR1; e) the cells of Escherichia coli are transformed by the prepared ligase mixture and clones, containing recombinant plasmid pPIC9HAblL-2 are selected.